Intravascular Location of Breast Cancer Cells after Spontaneous Metastasis to the Lung

Abstract

In this study, we examined the hypothesis that early pulmonary metastases form within the vasculature. We introduced primary tumors in immunocompromised mice by subcutaneous injection of murine breast carcinoma cells (4T1) expressing green fluorescent protein. Isolated ventilated and perfused lungs from these mice were examined at various times after tumor formation by fluorescent microscopy. The vasculature was visualized by counterstaining with 1,1-dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine (DiI)-acetylated low-density lipoprotein. These experiments showed that metastatic cells derived by spontaneous metastases were intravascular, and that early colony formation was intravascular. The location of the tumor cells was confirmed by deconvolution analysis. This work extends our previous study 1 We recently reported direct observations of the sequence of events in pulmonary metastasis after injection of fluorescent tumor cells into the venous circulation using fluorescent microscopy of isolated perfused lungs. 1 Because endothelium has receptors for oxidized or acetylated low-density lipoprotein (LDL), the pulmonary endothelium can be precisely visualized in these preparations through infusion of DiI-acetylated LDL. Because the lung is translucent, these methods allow high-resolution imaging of the microvasculature of the lungs at up to 100-m depth beneath the pleural surface and enable the precise localization of the tumor cells. We used these methods to show in a mouse experimental metastasis model that tumor cells attach to the pulmonary endothelium and proliferate intravascularly.We have also demonstrated that differences in apoptosis of tumor cells in vivo correlated to differences in metastatic potential. 1-3 Based on these observations, we proposed a new model for pulmonary metastasis in which endothelium-attached tumor cells that survived the initial apoptotic stimuli proliferate intravascularly. A principal tenet of this new model is that extravasation of tumor cells is not a prerequisite for metastatic foci formation. [1][2][3] Because the initial experiments were based on intravenous injection of tumor cells, it was possible that the results were an artifact of this mode of introduction of the tumor cells or of the relatively large numbers of cells infused simultaneously. To determine whether our observations in an experimental model of metastasis were valid for cells that metastasize spontaneously from a primary tumor, we have tested our hypothesis using a spontaneous metastasis model. Our previous results were obtained using fibrosarcoma cells, so we sought to extend this hypothesis to carcinoma cells by using the breast carcinoma cell line 4T1. 4 The results indeed indicate that extravasation is also rare after spontaneous metastasis. These observations confirmed that metastasizing tumor cells proliferate intravascularly within the lung. This suggests that drug discovery efforts could be directed to blocking the metastatic cascade at the steps of the initial Supported by the Susan G. Komen Bre...

Deficiency in surface expression of E‐selectin ligand promotes lung colonization in a mouse model of breast cancer

“…Tumor cells from the sLe xpositive variant were observed growing as small clusters in a linear fashion within capillaries. This is in accordance with the results of Wong et al, 45 who demonstrated similar intravascular growth in spontaneous metastasis of the murine 4T1 cell line. Tumor cells from the sLe x -negative variant were observed within capillaries as single cells adjacent to endothelium or as loose clusters of 2-3 cells without strong cell-cell interaction, and linear growth within capillaries was not observed.…”

“…In these areas, individual tumor cells appeared to be polarized with respect to the Using 4T1 cells as a spontaneous pulmonary metastasis model, it was shown that extravasation was not a prerequisite for metastatic growth. 45 The authors showed that tumor cells proliferated in blood vessels and then proliferating tumor cells massively extravasated via outgrowing the blood vessel. In our model, the differences observed in the morphology of growth patterns in the primary tumor suggest that modification in a carbohydrate epitope and a subsequent alteration in cell-cell adhesion might impact the pattern of micrometastases.…”

Semliki Forest virus biodistribution in tumor-free and 4T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon

“…4T1 is a mouse transplantable tumor cell line that is highly tumorigenic and invasive. 28 In contrast to previous studies, we have demonstrated that reduced dosage of recSFV provides preferential SFV infection in 4T1 tumor nodules, whereas the dosage increase contributes to broad dissemination of infection in the organism. Moreover, for the first time, the biodistribution of naked SFV recRNA in tumor-free and tumor-bearing mice was investigated on i.v., i.p.…”

Inhibition of pulmonary metastasis in a human MT3 breast cancer xenograft model by dual liposomes preventing intravasal fibrin clot formation

“…Several studies showed that an impaired platelet aggregation or a reduced platelet number results in the reduction of metastasis [35]. Our data suggest a direct role of platelets in metastasis in the MT3 model, because the into the surrounding tissue [36]. Additionally, they performed experiments using a spontaneous metastasis model and different cell types [15,31] to confirm these data [36].…”

“…It was also shown by the group of Muschel that murine 4T1 breast cancer cells metastasize as single cells to the mouse lungs in a spontaneous metastasis model [36].…”